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Decoding a cancer-relevant splicing decision

September 2018. Splicing is an important step in the maturation of newly made precursor messenger RNA transcripts. It comprises the removal of introns and joining of exons. Alternative splicing occurs naturally in humans and many other organisms and greatly increases the diversity of proteins that can be encoded by a genome. During alternative splicing, certain exons can be either included or excluded, thus leading to different transcript isoforms.

However, mutations that cause aberrant splicing are frequently implicated in human diseases such as cancer. Despite many efforts to understand the highly complex molecular rules of splicing, knowledge about regulatory elements and trans-acting factors is still far from complete. Splicing is catalysed by a multi-subunit complex called the spliceosome. Recognition of multiple regulatory elements by RNA-binding proteins guides the spliceosome and determines the splicing decision at each alternative exon. The information contained in the pre-mRNA sequence and how it is interpreted by RNA-binding proteins is known as the splicing code.

A team of scientists from the Goethe University Frankfurt, the Institute of Molecular Biology in Mainz and the University of Lisbon has established a new mutagenesis screening approach that provides insights into the splicing effects of mutations in humans and the mechanisms of alternative splicing regulation in general.

The method involves a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enabled the scientists to identify more than 1000 mutations affecting RON exon 11 skipping, which results in the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations.

The study also shows that heterogeneous nuclear ribonucleoprotein H (HNRNPH) is a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, the scientists pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11.

These results provide important insights into splicing regulation and the impact of mutations on alternative splicing in cancer.